The long-term goal of this project is to develop next-generation proteasome inhibitors that will serve as effective therapies 1) for multiple myeloma (MM) patients refractory to the existing proteasome-targeting drugs bortezomib and carfilzomib as well as 2) for patients with solid cancers. In the last decade, the FDA has approved the proteasome-targeting drugs, bortezomib (Velcade(R)) and carfilzomib (Kyprolis(R)) for the treatment of MM patients. These agents have greatly improved the prognosis of MM and firmly validated the proteasome as an important anticancer target. However, many patients eventually develop resistance after prolonged proteasome inhibitor therapy and few effective treatment options exist for such patients. In addition, the potential of proteasome-targeting drugs remain untapped for the treatment of solid cancers, despite their promising anticancer activities in preclinical models of solid cancers. Thus, it is critically important to develop novel proteasome inhibitors which can overcome the limitations of existing proteasome inhibitors and deliver the therapeutic benefits of proteasome inhibitor therapy to cancer patients of all types including solid cancers. We hypothesize that a new class of proteasome inhibitors with non-peptide scaffolds will provide new therapeutic options for MM patients refractory to bortezomib or carfilzomib and solid cancer patients. This is based on the drawbacks arising from the structural features shared among bortezomib, carfilzomib and nearly all proteasome inhibitors in drug development pipelines. Specifically, the structural features of peptide backbones and reactive pharmacophores contribute to a high susceptibility to chemical/metabolic inactivation and off-target interactions causing unwanted toxicities and an inability to achieve sufficient drug penetration within solid cancer sites. Using step-wise screening approaches combining virtual screen and in vitro enzyme kinetics, we identified several promising hits with diverse non-peptide scaffolds and proteasome inhibitory potencies. G4-1, a 1,3,5-substitutive pyrazole derivative is our current lead candidate, displaying its anticancer activity in multiple cancer cel lines, including cell lines resistant to bortezomib or carfilzomib. In addition to having excellent metabolic stability, G4-1 displayed promising anticancer efficacy and safety profiles in vivo. With the goal of further improving the potency and other pharmacokinetic properties of G4-1 to be suitable for oral dosing, we propose the following aims: 1) to extend our lead optimization efforts in developing more potent proteasome inhibitors with improved drug-like properties, 2) to investigate the cytotoxic mechanism of action of optimized non-peptide proteasome inhibitors, and 3) to evaluate the in vivo anticancer efficacy, safety and pharmacokinetic profiles of selected lead non-peptide proteasome inhibitors. Successful completion of the proposed work will yield novel non-peptide proteasome inhibitors that can advance to next stages of drug development efforts and ultimately lead to effective therapies for patients with MM and solid cancers.